Forward

The blood brain barrier is a physical barrier composed of endothelial cells that are connected
by tight junctions. It is created at the interface between the central nervous system and the
blood circulation at the brain microvascular level.

BBB plays a major role in maintaining the neural function and synaptic content through ions
regulation. It acts as a protective barrier that prevent macromolecules and toxins from
entering the CNS. This function of the BBB become an obstacle for treatment of brain
conditions, the drug therapies unable to pass the BBB and leads tofailure to exert their
therapeutic effect on the brain.

Researchers have been started directing their research area on finding a model way of treating
brain diseases with the fact that BBB has a great investment in optimal drug delivery to the
brain. As a group working on this literature review we will do a review of the ways
experienced and direct our report on one the overviews about the drug delivery to the brain.
Function of the BBB:

The Main function of the BBB is the protection from exogenous as well as endogenous
compounds such as toxins or unwanted metabolites to prevent any damage happening in the
brain. It is known to have the largest surface area for exchange between the brain and the
blood. The BBB also plays a role in maintaining homeostasis for the cerebral nervous system
to prevent any malfunctions. (Bernacki J et al., 2008)

In the figure above illustrates the barriers found in the brain as [A-B-C] (N. Joan Abbott et
al., 2010)

Structure of the BBB:

The BBB structural basis are brain micro vessels. The structural unit of the BBB is a
neurovascular unit which composed of 4 cell types:
- Astrocytes
- Neurons
- Pericytes

Brain microvascular endothelial cells (BMVEC) which is the key element for BBB because:

1- Endothelial cells are connected to each other by tight junctions and they limit the
paracelullar diffusion by sealing the spaces between the endothelial cells.
2- For the brain to get its high energy and nutrient needs, there is an influx\efflux
transport and carrier system on the surface of the (BMVEC), as an example; solute
carrier (SLC) and ATP binding cassette (ABC) they will allow the brain uptake of
nutrients from the blood (such as glucose, amino acids and ions) and they also excrete
 wastes and keep neurotoxic substances from passing to the brain. There are side
effects to these transporters by effecting the therapeutic efficacy of the drugs by
blocking these from reaching the site of action which would lead to failure of the
treatment of many brain diseases.

On the other hand, the other cell types (Pericytes, Astrocytes and Neurons) have major
functions as well:
- They enhance the formation of the tight junctions by releasing certain substances
- They lead to a functional barrier by expressing the efflux transporters

In the figure above illustrates the anatomy of the Neurovascular unit/blood-brain barrier that
consists of brain capillary endothelial cell, pericyte, astrocyte, and neuron.(N. Joan Abbott et
al., 2010)
Inter-endothelial junctions:

The key structures of BBB are BMVEC and inter-endothelial junctions which link the
BMVECs together. There are two types of inter-endothelial junctions: tight junctions TJs and
adherence junctions AJs.

Tight Junctions:

Compose of complex proteins that fills the space between endothelial cells allowing small
molecules like water and ions to pass and protecting brain from macromolecules and
neurotoxins. These proteins are occludin, claudins which are linked to regulatory proteins
ZO-1,ZO-2,ZO-3 and cingulin, in addition to junctional adhesion molecules JAMs. Tight
junctions are created at the apical side of BMVECs and serves in linking them.

Adherence Junctions:

Located under the tight junctions, compose of vascular endothelialcadherin proteins that are
linked to the cytoplasm through alpha, beta and gamma catenin scaffolding cytoplasmic
proteins like Beta catenin, alpha actinin and vinculin which act together to support the tissue
membrane. Any disruption of these junctions will cause barrier disruption.
Routes of transport across the blood brain barrier

The diagram above illustrates the rout of transport across the BBB [Passive diffusion, ABC
transporter efflux, Solute carrier SLC, Transcytosis receptor mediated-Adsorptive mediated
and mononuclear cell migration] where the passive diffusion mostly accurse to high lipid
soluble substances. ABC transporters, which mainly focus on the efflux of substances
comparing to the passive diffusion and are one of the major causes of MDR. Solute carrier
SLC, may work as passive or secondary or primary active which transports many essential
polar molecules such as glucose, amino acids and nucleosides in to the CNS. Transcytosis
receptor mediated, these transporters transport macromolecules such as peptides and proteins
across the cerebral endothelium, However Adsorptive mediated transcytosis
transportpositively charged macromolecules and can also transport across the endothelium,
both of them carry the macromolecules as vascular-based systems. Mononuclear cell
migration, Leukocytes cross the BBB either being inactivated and get activated inside or
activated before entry by a process of diapedesis through the endothelial cells (penetrating
close to the tight junctional regions), or via modified tight junctions.(N. Joan Abbott et al.,
2010)

Drug transporters and the structural features of the BBB in normal state.

Knowing that the tight junctions has a major role in drug resistance and also blocks the entry
of foreign materials, in fact lipid soluble substances and substances that are authorized to
enter the transport system can cross the BBB. The influx transport plays an expert role in the
uptake of nutrients needed for the BBB in order to have the high nutrient level and the energy
demand, whilst the efflux transporters assure that there are no wastes available in the BBB
including pathogens. In order for these mechanisms to proceed, there are a number of
transport and carrier systems such as the Adenosine triphosphate-Binding Cassette (ABC)
transporters and the SoLute Carrier Family (SLC) take place, The Adenosine triphosphate-
Binding Cassette (ABC) transporters have an essential role in blocking neurotoxic substances
reaching the brain which results in transporting the harmful materials outside the brain, while
the SoLute Carrier Family which are a group of transport proteins have a professional role In
controlling the cellular uptake and the efflux of nutrients, neurotransmitters, metabolites and
toxins.

ABC Transporters:

ABC transporters can be found in many organs like (liver, kidney, GIT,brain and other), they
allow the crossing of lipid soluble substances by hydrolyzing and binding the ATP. They are
transmembrane proteins found at the luminal and abluminal plasma membrane of BMVECS,
therefore ABC plus the tight junction will have a significant barrier function of the
neurovascular unit (NVU).

There are 50 ABC known in the human body, and they are divided into seven subfamilies and
this division was made according to their structure similarities.
Among these seven families are: ABCB also known as (MDR, Multidrug Resistance Protein)
ABCC or (MRP, Multidrug Resistance-associated Protein) ABCCA, ABCD and ABCG2 or
(BRCP, Breast Cancer Resistance Protein).However at the BBB level there are specific type
of transporter that are known till now, which are responsible for the efflux transport, among
these are: (ABCB1, ABCC1,2,4,5,6 and ABCG2).

SLC Transporters:

SLC are (Solute Carries Transporters) superfamily of membrane transport they consist of
about 300 membrane and are categorized into 43 subfamilies.
Among these families there are two transporters that are mainly found in the brain, and have
an important role in the brain, in transporting into and out of the BBB like: organic material,
nutrient, nutrient substances such as (glucose, amino acids, etc) and plays a role in efflux
transport, these two are (Organic Anion Transporting Polypeptide OATP/SLCO) and (Organic
Anion Transporters, OAT/SLC2A)
SLC or (OATP/SLCO) (OAT/SLC2A) they cooperate with MDR and MRP in order to
remove the xenobiotics from the BBB.

Transport at the Blood-Brain Barrier:

There are four basic mechanisms by which solutes move in both directions across the BBB,
and they are:
1-Passive Diffusion: and it has two main pathways depending on the size and the lipophilicity
of the molecule
A. Para cellular aqueous pathway
B. Trans cellular lipophilic pathway

2-Transport via transport protein

These transporters are like SLC and ABC they act sometimes as efflux transport and
transport essential polar molecules like glucose and amino acids out of the endothelium.

3-Transport via a receptor and it has two mechanisms which are endocytosis and transcytosis
and by having these two steps it gives an advantage to transport a macromolecule or protein
such insulin and transferrin.

4-Adsorptive Transcytosis is similar in method to Transport via receptor in that they have
both endocytosis and transcytosis, more than that they catonize the large protein molecule to
Increase their uptake through the BBB.
Drug Transporters

The BBB has a dynamic barrier function that is composed of structures control the movement
of molecules between CNS and blood as well as the barrier function of the brain. Brain
capillary endothelial cells of the vascular cerebrum, pericytes which cover the endothelial
cells, astrocytes available around capillaries of the brain and neurons which act as nerves for
the microvessels. These 4 cell structures types and function of the BBB known as the
neurovascular unit. The figure below shows the 4 cell types of the NVU.

Endothelial cells surrounding blood capillaries, pericytes surrounding endothelial cells,

astrocytes and neurons are the key components of the NVU.

Many factors affect NVU function as a barrier of molecules comes in and out from blood
to CNS. Tight junctions play a major role in making BBB a physical barrier for delivery of
therapeutic drugs to the brain by filling spaces between the adjacent endothelial cells
preventing water soluble molecules, ions and aminoacids diffusion between endothelial cells
from blood to the brain which will affect nutrients and energy delivery to the brain. To meet
the required levels of nutrients in the brain, ATP-binding cassette (ABC) work as a critical
delivery regulation device that affects directly the transport and barrier functions of the brain
by dual-action mechanism. ABC transporters work as influx transporters enhance glucose,
nutrients, aminoacids and ions passage from blood to the brain. On the other hand, ABC
transporters work as efflux transporter that decrease passage of Xenobiotics, toxic molecules
and wastes into the brain. Also, ABC transporters prevent large number of therapeutic drugs
from reaching brain and doing their action. By this from an impediment for CNS diseases
therapy.

Therefore, researchers in this field of interest aim to untangle signaling mechanism

intracellularly that interfere passage of therapeutic drug molecules through the CNS, by
modulation of ABC transporters to allow therapeutic drug delivery to the brain and CNS by
controlling up-regulation and down-regulation of molecules movement between BBB and
blood.
An example on this approach is that down regulation of ABC transporters on the BBB
enhances therapeutic drug delivery while up-regulation of ABC transporters increases
protection function of the brain against side effects of CNS diseases therapies peripherally.
Recently, it has been approved that ABC transporters available in NVU and brain cells has a
direct effect on the brain and CNS diseases. Therefore, good understanding of ABC
transporters physiology and pathophysiology allows modulation of the ideal way to enhance
therapeutic drug delivery across the BBB into the brain.

ABC transporters

ATP-binding cassette (ABC) transporter is one of the earliest and most common
transmembrane protein that controls active transport of lipid molecules, steroids and
therapeutic drugs against the concentration gradient requiring energy.

Two classification of ABC transporters, nucleotide-binding(NBD) and transmembrane

domains(TMD) and to be functional, ABC transporters need at least 2 of each NBD and
TMD. TMD forms ligand binding site and to give ligand-specificity. NBD facilitate diffusion
of ATP through the membrane(Loo, 2002). Therefore, in order to achieve ideal function and
transport of ABC transporters, each family of ABC transporters contain NBD with
homologous characteristics such as walker A & B motifs, ABC signature and staking
aromatic D,H and Q loops that are special for each(Linton, 2007). ABCD1 form homodimer
in itself and heterodimerize with ABCD2 and ABCD3(Liu, 1999). ABCG1, 4,5 and 8
heterodimerize while ABCG2 hemodimerizes(Cserepes et al.,2004; Graf et al., 2003;
Kage.,2002). Each of ABCD1, ABCG1,2,4,5 and 8 should contain one NBD and one TMD
where they both have to dimerize in order to achieve full function activity.

ABCAFamily

AmongallABCtransportersfamiliesintheCNS,mostresearchisdoneonABCA1family
whichhasbeenfoundinmanyproteinsofthebrainaswellasintheepithelialcells,
endothelialcells,astrocytesandneurons(Koldamova et al.,2003;Fujiyoshi et al.,2007; Kim et
al.,2006)(Figure)

FigureABC Transporters in the CNS. ABC A: yellow, ABC B: red, ABC C: blue, ABC D:
green, ABC G: magenta. For transport- ers shown in gray, cellular localization is not known.
Only trans- porters that were detected at the protein level are shown(Hartzetal.,2011).

ABCA1has been found in abluminal membrane of brain capillary endothelium of the

pig(Panzenboeck et al., 2002),also it has been found in the rat choroid plexus cell line, TR-
CSFB3 (Fujiyoshi et al., 2007),whileABCA2-9 has been found in the mRNA and protein
level of the brain capillary endothelial cells of human, rat, mouse(Gosselet et al.,
2009,Ohtsuki et al., 2004Warren et al., 2009 Quezada et al., 2008),astrocytes(Kim et
al.,2006),microglia(Kim et al.,2006), neurons(Kim et al.,2006; Wellington et al.,2002), oli-
godendrocytes (Zhou et al.,2001) and choroid plexus epithelial cells(Fujiyoshi et
al.,2007;Bhongsatiern et al.,2005; Matsumoto et al.,2003).

ManyresearcheshavebeendoneonABCAtransporters,butwedonthaveenough
informationabouttheirfunctionintheCNS.IthasbeenfoundthatABCAtransportersis
relatedtoapolipoprotein-dependent cholesterol efflux(Gosselet et al.,2009) in which

1. it has a role in brain development

2. upregulated in astrocytes
3. cholesterol level in the cells decreased which is mainly because of ethanol effect
whilecholesterolproductioninsidethecellsdoesntchangebytheaffectofethanol.
4. Ithasbeenfoundinneuronsandendothelialcellsofporcine(Panzenboeck2002
Minagawa2009.
5. IncreaseintheintracellularcholesterolaswellasthedecreaseinapoElipidatonwill
leadtodecreaseofcholesteroleffluxinastrocytesandmicogiliainthemicewhich
lackABCA1transporter Hirsch-Reinshagen,2004

Newstudieshavebeendoneonastrocytesofmanyspecies.Invitrostudiesonhumanfetous
showedthatethanolincreasestheABCA,alsoinvivostudyhasbeendoneonmouse
(Guizzetti et al.,2007).Also,ithasbeendetectedthatABCAtransportershavearoleinlipid
metabolismandsterolhemostasis(Gosselet et al.,2009;Hirsch-Reinshagen et al.,2004)

IthasbeensuggestedthattheremightbearelationbetweenABCA1andAlzheimersdisease
(AD)asthereductioninapoElipidationwillleadtodecreasedamyloidBproteolytic
breakdown,whichwillresultinincreasedriskofdevelopingAD (Wahrle2004)

ABCBFamily

MoststudiesdoneonABCtransportersintheCNSwheremainlyfocusinginABCB1
transportersandfewresearchattemptshavebeendoneonothermembersofthisfamily.
Capillaryendothelialcellsinhuman,cat,cow,pig,mousewherefoundtohavemRNAfor

Phosphatidylcholine transporter, ABCB4 (P- glycoprotein 3, PGY3), and the

mitochondrial transporters, ABCB6 (mitochondrial ABC transporter 3, MTABC3),
ABCB7, ABCB8 (MABC1), and ABCB10 (mitochondrial ABC transporter 2,
MTABCT2)(Warren2009).
bile salt export pump, BSEP(ABCB4andABCB10)inHumanandratchoroidplexus
(Niehof2009 Choudhuri2003)

SinceinformationfromresearchandstudiesonmembersABCB211inABCBtransporters
familyarelimited,thefocusisonABCB1transportersinCNS.ThatsbecauseABCB211
havenotbeenfoundintheproteinlevelofthebrain.

ABCB1(Pglycoprotein,Pgp,MDR1)

ABCB1Expression,Localization,andFunction

In1976julianoandlinghavediscoveredABCB1inadrugresistantwhichexplainedthe
conceptofmultidrugresistanceincancerdisease (Juliano1976).In1980sithasbeenfound
thatABCB1arepresentinthehighperfusionbarriertissuesofkidney,liverandintestine
Thiebaut1987.In1989,ithasbeenfoundABCB1protiensinbraincapillaryofendothelial
cellsinhuman(Thiebaut1989 Cordon-Cardo1989).Lateron,withinsomeyears
ABCB1inmRNAandinproteinlevelhasbeenfoundfromtheisolationofmonkey,dog,
rat,mouse,cow,dogfishandkillifish.Also,inendothelialcellsfrommonkey,dogandcat
(Samoto1994 Cordon-Cardo1989).

Inthepast15years,ithasbeenfoundthatABCB1transportershavegreatroleinbloodbrain
barrierfunction.ButtherearefourreasonsforABCB1toactasbarrierpreventingpassageof
xenobiotic,toxinsandmanydrugswhichareexpression, localization, potency, and multi-
specificity Begley2004

1. ABCB1 are found upregulated in the brain capillary membranes. Results have been
showed that ABCB1 are markedly increased in ex-vivo experiment for rat or mouse
brain capillaries more than any plasma membranes tested from any other tissues
(Miller2008Bauer2005)

2. ABCB1 has the advantage over other transporters families as a CNS barrier Figure2
and as a drug efflux pump for expressing in the luminal membrane of capillary
endothelial cells. It has been agreed on that in this kind of research, since its
approved by clear clues that explain localization of ABCB1 in the luminal plasma
membrane Bauer2006 Tsuji1992.

3. ABCB1 has been found in a variety of molecules with different diversities, ranging
from small molecular weight molecules like morphine, verapamil, and loperamide
(MW 285-477 Da) to small molecules like peptides such as amyloid-beta (MW 4200
Da)( Hartz2010Sugawara1990 Tsuji1992 Kuhnke2007 Tanaka1994 Beaulieu1997
Biegel1995 Shirai1994).Also,ABC1hasbeenfoundtohavexenobioticsofmany
classeslikechemotherapeutics, HIV protease inhibitors, opioids, antibiotics
Bauer2005

ABCB1 Regulation:

The focus was largely done on ABCB1 regulation mostly in the brain capillary cells and to a
lesser degree in astrocytes and the choroid plexus. This also includes the recent identification
of several signaling pathway related to the regulation of ABCB1 in the brain. In this part we
will mainly discuss the summary of finding of ABCB1 regulation through inflammatory
mediators, oxidative stress and nuclear receptors. (Hartz, A et al., 2009)

ABCB1 regulation in inflammation:

One of the main researches done was identifying the effect of inflammation on the ABCB1
transporter. A study was done by using isolated rat brain capillary, exposing it to these pro-
inflammatory mediators such as TNF, LPS or ET-1 showed a direct reduction which as
reversible of ABC1 transport activity, however there was no change in the protein expression.
This mechanism happens by the binding of these pro-inflammatory mediators to the receptors
leads to the activation of NOS and PKC and causes rapid retrieval of ABCB1 from the
membrane and the decrease of ABCB1 mediated transport. In fact the exposure of
inflammatory mediators for a long period of time causes increase expression of ABCB1
protein and transport activity. The long term study had the same signaling pathway as the
short term. Other researches were done by using vitro cultures of human or pig brain capillary
endothelial cells and showed an increase of ABCB1 mRNA including the protein levels after
the TNF-Alpha treatment however there was no effect on the function of ABCB1. When done
in vivo studies the result was conflicting. Seelbach et al. has done the study by the use of
peripheral inflammation rat model and showed up regulation of ABCB1 protein expression in
the brain capillaries and this had led to the reduction of brain uptake of weak ABCB1
substrate such as morphine which eventually due to the up regulation of the transporters,
prevent the entry of morphine to the brain. (Hartz, A. 2004)
In these studies it has been shown that inflammatory mediators play a major role on the effect
of ABCB1 in the brain and since inflammation is a complex process and is dose, time,
context and model dependent. (Hartz, A et al., 2006)

ABCB1 regulation in oxidative stress:

Known as the Reactive oxygen species commonly accurse during CNS inflammation and
mostly in CNS disease such as stroke, brain tumor, epilepsy, trauma multiple sclerosis,
Parkinsons and Alzheimer diseases. A Study was done using rat brain capillary endothelial
cells in vitro by exposure of hydrogen peroxide for 1 to 2 days resulted in increase of ABCB1
mRNA and protein expression including vincristine transport. Moreover, depletion of GSH in
rat brain capillary endothelial cells in vitro causes up regulation of ABCB1 mRNA and
protein expression well, but the response is reversed or inhibited by ROS scavenger known as
M-acetlycysteine. Therefore the reduction of GSH causes increase in ROS which eventually
causes increase in ABCB1. These studies could be an advantage for stroke research since at
this condition there are high levels of these signals. (Felix, R. et al., 2002)

ABCB1 regulation in nuclear receptor:

Nuclear receptors are known as the transcription factors; once they bind to a ligand they
cause activation of the targeted gene. They control the transcription of ABC transporters such
as ABCB1, ABCC2 and ABCC3 including metabolizing enzyme, and are activated by
xenobiotics. Since they affect the ABC transporters this leads to the effect of
pharmacokinetics of many drugs and therefore is a concern clinically. (Perloff, M. D et al.,
2007)

ABC C Transporters

ABC C family contains 13 members and 9 out of them are transporters ABCC1, 2, 3,4,5,6
and ABCC10, 11, 12. However not all of them are well studied, for example there is no
information about transporter ABCC12 in the CNS while for ABCC10 it has only been seen
at the mRNA level in the CNS. However ABCC1, 2, 3, 4, 5 are well studied and do contain
information there therefore will be focused more on them. (Zhang, Y., 2000)

Introduction of ABCC1

Mostly found in astrocytes, microglia and choroid plexus of the human brain, their function
study was tested by using rats astrocyte cultures, noticed during oxidative stress a high
release of GSH caused by the transporters. More studies must be done regarding their
function. (Saito, T et al 2001)
Regulation of ABCC1

There are two mechanisms that could explain the regulation of ABBC1 transporters starting
with the up regulation of these transporters including ABCB1 during HIV mainly caused by
the proteins of this virus tat and gp120. This was noticed in bovine brain capillaryendothelial
cells and in mouse and rat astrocyte cultures. Inflammatory mediators do as well play a role
in this up regulation such as the TNF, MAPK, JNK and NF. This mainly happens because the
HIV virus targets the microglia and the astrocytes in the brain, therefore it is important to
under the mechanism. (Hayashi, K et al., 2005)
Another mechanism of regulation of ABCC1 transporter is the unconjugated bilirubin that
causes the up-regulation of these transporters and promotesits trafficking from the Golgi
apparatus to the plasmamembrane, this is said to be as a defense mechanism for bilirubin
against its own cytotoxicity indisease states such as hepatitis,which therefore causes the up-
regulation of bilirubin levels that causes jaundice. However the exact mechanism is unknown
for the up-regulation of ABCC1 caused by bilirubin but could be due to the activation of the
nuclear receptors. (Gennuso, F et al ., 2004)

ABCC2 and ABCC3 transporters

There is no enough information that demonstrates the location, mechanism and regulation of
these transporters in human cell.(Bauer, B et al ., 2008) . However tests have been done on rat brain
capillaries and suggest their existence in the endothelial cells and choroid plexus. Further
investigation need to happen regarding these transporters. (Poller, B et al .,2008)

ABCC4 transporters

ABCC4 transporter is said to resemble ABCB1 in structure, these transporters are found in
luminal membrane of brain capillaries in the human brain, however their function have not
been approved yet. In fact ABCC4 transporters play a role in mediating efflux transport of
organic anions, glutathione-, sulphate-, or glucuronate-conjugated drugs, prostaglandins, and
nucleoside analogs, including the efflux of methotrexate, topotecan, and thiopurines which
could explain their function of protecting the blood brain barrier from xenobiotics, as this
leads to drug resistance. However, their importance in the BBB is not well known.
ABCC4 mRNA expression are also found in choroid plexus of the human brain. A study was
done in wild type and ABCC4 knockout mice explained the defensive effect of this
transporter to the brain by preventing topotecan penetration into the cerebral spinal fluid.
Last but not least, the expression of these transporters has been reported in other brain cells,
ABCC4 protein was discovered in astrocytes of the human brain. However further studies is
needed in their location including their function. (Leggas, M et al ., 2004)

ABCC5 Transporters

ABCC5 transporters are studied the most in the CNS, researches demonstrate the presence of
these transporters expressed at the mRNA level in brain capillaries or cultured brain capillary
of the endothelial cells from human, cow, pig, rat, mouse. These transporters showed efflux
effect on methotrexate in the human brain capillary endothelial cell line. There are no further
function of this transporters identified.
Further locations of ABCC5
ABCC5 mRNA and protein where found in astrocytes of the human brain slices.
The expression of ABCC5 protein found in neurons of human brain slices.
 ABCC5 mRNA in human rat choroid plexus.

However their functions where not identified and need further studies. (Hirrlinger, J et al .,
2002)

ABC D Transporter

ABCD transporters transport fatty acid into the peroxisomes for metabolic breakdown,
located in the peroxisomal membrane. Specifically ABCD1, transports very-long-chain
fattyacids and fatty acyl-CoAs. However, it is a half transporter which it order for it to be a
completely functional it has to dimerize with ABCD2 or ABCD3. (Theodoulou, F et al ., 2006)

Any mutational defects of the ABCD1 gene could possibly cause a fatal condition known as
drenoleukodystrophy, this accurse due to the transport protein inability to transport very long
chain fatty acids into peroxisomes for metabolic breakdown. This event causes accumulation
of the fatty acids in the brain and leads to BBB breakdown and a progressive destabilization
of the axon myelin sheaths which eventually causes death. (Moser, H et al ., 1995)

ABCG Transporters

There are five transporters that have been studied well among the ABCG family ABCG1, 2,
4, 5 and 8. Starting with ABCG 1, 4, 5, 8, their main role is to transport cholesterol, sterols,
and biosynthetic sterol intermediates and isalso responsible for cholesterol and sterol
homeostasis. (Tarr, P et al., 2008)
Moving on to ABCG2, the transporter was discovered due to its strong resistance to
mitoxantrone which was nonresistant to ABCB1 and ABCC. Located at the human neural
stem cells localized to the plasma membrane and has been shown to potentiate prazosin
transport, which has been suggested probably as a defense mechanism against xenobiotics to
protect the stem cells. (Lee, G ., 2007)
In fact, Dauchy et al. has noticed due to the high level of mRNA and protein levels of the
transporters ABCG2 and ABCB1, they were the two main ABC transporters expressed in
isolated brain capillaries from human cortex biopsies, having 20-25 folds of these
transporters in the capillaries and cortex tissues. (Cisternino, S et al ., 2004)
Another important fact is that ABCG2 is responsible for the efflux of chemotherapeutic drugs
such as imatinib, dasatinib, and lapatinib, therefore makes it difficult in treating brain tumors,
especially glioblastoma. (Legas. J et al., 2009)

SLC Transporters

SoLute Carrier (SLC) are located almost around the body with over 300 members classified
into 43 families, among these families Organic Anion Transporting Polypeptides
(OATP/SLCO) and Organic Anion Transporters (OAT/SLC2A) are said to be found in the
BBB, these SLC transporters support the brain by transporting nutrients that the brain needs,
since TJ block these substances from entering as glucose and amino acids, and play a major
role in transporting organic compounds to the BBB.
In fact, these transporters also have a major role in the efflux transport for the removal of
xenobiotics from the brain which leads to MDRs. However, a further study regarding these
transporters is not published yet. (Harati R., 2012)

Pericytes:

Pericyte secretes growth factors and extracellular matrix components which help in
maturation and maintenance of BBB as well providing physical support to the EC, there are
different types of pericytes accompanied with different blood vessels and the CNS has shown
that it contains the highest numbers of pericytes. In vitro models there are two types co-
culture and tri-culture, for the co-culture we used BBB-ECs and astrocytes and when we
added pericytes it became triculture system, it enhanced the system and organized it in to
stable capillary-like structures showing us the importance of pericytes in forming and
maintains the vasclature of the BBB. other factors which are secreted from the pericyte are
transforming growth factor (TGF)-B, angiopoieten, platelet-derived growth factor (PDGF)-B
and sphingosine-1-phosphate (S1P).

Pericytes reduce BBB permeability through N-cadherin-dependant binding to EC, this

binding promotes vascular maturation and integrity. Pericytes support cerebral blood flow in
experimental models of ischemia and brain tumors through induction of angiogenesis.

After all these function of pericytes that we have mentioned above, the exact function of
pericyte is still under investigation. (R.Balabanov, et al 1998)
Astrocytes:

Are considered inducers of the BBB properties. It covers 99% of the brain and there is a thin
layer of compact BM separating the two layers. Astrocytes-indothelial cells are known to
modulate inflammation responses in the CNS by interactions that regulate many factors such
as EC proliferation, angiogenesis, transporter protein expression, TJs protein expression,TJs
morphology (A.Prat,et al 2001).
In vitro models of isolated and cultured BBB-ECs they lose their BBB features such as p-
glycoprotien and TfR expression. However we can reinstate by co culturing BBB-ECs and
astrocyte or astrocyte conditioned media (ACM). These evidences show us the importance of
astrocytes and the factors that are needed for BBB function, these factors should be soluable
however the exact identity of sloubale factors are still unkown but there are candidate such as
fibroblast growth factor (FGF), glia-derived neurotropic factor (GDNF), TGF-B, SRC-
supressed C kinase substrate (SSeCKS), meteorin, angiotensinogen and hedgehog family.
(K.Wosik, et al 2007)
Base membrane:
The BM is composed of several extra cellular matrix including laminin, fibronectin, collagen
type IV and heparin sulphate proteoglycan, dystroglycan, and it is found between the BBB-
ECs and the peri-vascular cells.

BM has two types of membranes and this was found by doing yltra structure and
immunofluorescent test that demonstrated these two membranes are close with brain micro
vasculature and these membranes are called endothelial and parenchymal BM.

When the BM attach to the NVU they form a substrate for cellular differentiation and gene
expression so the main function of BM as a tissue scaffold. Endothelial, astrocyte, lesser
degree of pericyte derived extracellular matrix proteins are important constitute of glial
matrix and they enhance the BBB integrity. Paracrine potency of biological active molecules
and local concentrations can be increased by soluble factors that can be secreted by glial cells
which can be captured by ECM proteins depending on their charge. For EC adhesion we need
BB-ECs to express a number of integrins to the basal lamina and any alteration in the
expression of BBB basal lamina or the integrins there will be a relation in BBB breakdown.
(B.T.Hawkins, et al 2005)
Adherent and tight junctions: molecular fences and dynamic signaling regulators of BBB
integrity.
Large molecular complexes are sealed due to the intercellular spaces of tight junctions and
impermeable biological barriers are called tight junctions. Large multi protein complexes are
one of the main compositions of tight junctions, they act as molecular fences by meditating
close intercellular contact between the adjacent cells. There are three types trans-membrane
protein which are the main tight junctions constituent. (A.W.Vorbrodt, et al 2003)
1. Junctional Adhesion molecule
2. Occludin
3. Claudins

The exclusion of blood-borne from CNS selectively occurs by extracellular loops of these
protein by forming a paracellular barrier. Mice can have proper tight junctions functioning
(T.Nitta,et al 2003), in Occludin deficiency which means the structural integrity of the tight
junctions are not requiring Occludins in their structure, while the Claudins are important
constituent of the maintenance of BBB
The extracellular loops of Homophillic and Heterophillic bindings are closely sealed due to
Occludins and Claudins. The expression of Claudins-3 and Claudins-5 in brain-ECs and their
significant role in vessel angiogenesis and permeability.
The promotion of Cell to cell contact in Homophillic coordination by intercellular tight
junctions of Murine and human CNS-ECs due to JAM-A localization but its still unclear how
they can restrict the BBB permeability and also the application of JAM-A in angiogenesis,
leukocyte tran-migration and as a receptor for viruses. JAM-B and JAM-C have been found
in Murine and Purcine CNS-ECs but still the role unclear.
The link between actin microfilaments and tight junctions proteins is mediated due to a
cytoplasmic region which contains high number of adapter proteins which is responsible for
the link mediation and its located under tight junctions and membrane proteins.
The maintenance and the regulation of a wide signaling pathways is important for EC
survival and tight junction assembly by the interaction of different structural proteins and
JAM-A, Occludins and Claudins.
Bridging and immobilization of membrane-anchored tight junctions proteins with epithelial
barrier and actinfilaments in EC is by membrane-associated guanylate kinase inverted
proteins (MAGI-1-3) (L.Guillemot, et al 2008) and zonaoccludins (ZO-1,-2 and -3) which
they are a significant and important adapter proteins.
Numerous protein protein interaction domains such as SH3 and PDZ are contained in these
adapter proteins. The formation of signaling scaffold due to the interaction to these domains
with the adapter proteins and cytoplasmic tail of integrinal membrane proteins.
The maintenecne and biogenesis of tight junctions needs the involvement of other
intracellular proteins such as: AF-6, CINGULIN, Calcium+2-Dependant serine protein kinase
(CASK) (E.Dejana, et al 2008) and 7H6 antigen and other signaling molecules such as RhoA
and Rac.

AJs work in endothelium and epithelium when there is low permeability of the BBB and
BCB, they consist of trans membrane protienscadherins and cytoplasmic protienscatenins so
they regulate the para cellular permeability and because the AJs are homophilic and there are
interactions between the extracellular domains of calcium dependantcadherins on the surface
of adjacent cells they give the properties of adhesion while catenins work as linker and
support the formation of adhesive contact between cells. In ECs there are the highest levels of
vascular endothelial (VE)-cadherins also its known as cadherin-5. To compromise signaling,
regulatory and scaffold(E.Dejana, et al 2008) proteins we need first the formation of multi
molecucle complexes which can be formed by the clustering of VE-cadrehins at cell-cell
contact.
Structural features of the BBB in disease state

The abnormalities of tight junctions in human disease:

It has been shown that large number of cytokines play a major role in endothelial and
epithelial Tight junction function including the actin cytoskeleton, which mostly are IL-0, IL-
1, IL-3, IL-4 tumor necrosis factor alpha (TNF) and interferon gamma (IFN). Some
examples that effect the TJ function are when it comes to cancer diseases such as in Breast
cancer (invasive ductal cancer) effect the Claudin-1 and ZO-1 , Prostate cancer (prostatic
adenocarcinoma ) effect the Claudins-1 , -3, -4 and-7. As for Inflammation, Inflammation
bowel disease (Morbuscrohn) effect the claudins-2, -3 -5 and -8 including ZO-1 and so on.
These diseases are said to affect the TJ function by reducing its activity in their different
mechanisms. (C. Frster., 2008)

Cellular abnormalities caused by brain cancer

Endothelial cells:

When tumor takes place they start to secret factors and components in order to form new
vessels. Normally this happens by angiogenesis using an existing vessel while in the
endothelial cells the balance between endothelial cell division and apoptosis are shifted to
proliferation, But in general the growth of tumor vessels in the endothelial cells do have
similar features as the normal pathway.

Due to the lacking of tight endothelial monolayer of the tumor vessels, this result in leakiness
of the vessels and including the absence of lymphatic vessels as well as contractile stromal
elements, results to rise of interstitial fluid pressure, Because of those structural abnormalities
mentioned this effects the blood flow and interfere in the drug delivery.(Hashizume. H et al.,
2005)

The effect of ABC transporters in disease state:

Researchers of ABC transporters have noticed that these transporters play a major role in
many CNS diseases, including those of Parkinsons disease and HIV and so on, but there isnt
enough information regarding the ABC transporters in these diseases so the focus will be
mainly on the three CNS diseases (Brain cancer, epilepsy and Alzheimer disease) where the
ABC transporters can be mostly explained. (Hartz et al., 2011)
Brain Cancer:

The treatment of cancer in the brain is yet very difficult to reach comparing to the periphery.
The ABC drug efflux transporters which are ABCB1, ABCC1, 3, 5 and ABCG2 are found to
be expressed in large amounts and this leads to preventing the entry of chemotherapy in to the
CNS which would result in worsening the expression of cancer stem cells and the tumor will
grow larger after surgery. Therefore the presence of these transporters ABCB1, ABCC1 and
ABBCG2 have generated a huge barrier in the treatment of brain tumors. There have been 2
methods that were suggested that might solve the problem which are: ABC transporter
inhibition and ABC transporter modulation.(Hartz et al., 2011)

ABC transporter inhibition: It has been shown that in the vivo study of mice having an
intracerebally implanted human glioblastoma, the inhibition of ABCB1 with the selective
inhibitor PSC833 (valspodar), prior to treatment with the chemotherapeutic paclitaxel.
However, the combination of the 2 medications resulted in reduced tumor volume up to 90%,
comparing to paclitaxel given alone which had no change on the tumor size.The design of the
new ABC transporter inhibitors with dual blocking activity has proven to decrease in
glioblastoma growth time in mice, And another inhibitor known as NSC73306 that inhibits
ABCG2 and as well as destroys the over expression cancer cells of ABCB1.These studies are
not yet confirmed to be proven in effecting in brain cancer chemotherapy.

ABC transporter modulation: Another recent technique has been implemented to prevent the
ABC transporter-MDR is the targeting of the intracellular mechanisms that control these
transporters. The main achievement of this technique is to temporary turn off these
transporters in order to deliver the therapeutic drug into the brain and tumor tissue.(Elbling. L
et al., 2002)

Examples of ABC transporters in Disease state:

Transporter type: The disease Refernces

ABCB1 Brain Cancer (Bauer.B et al., 2008)
- regulation of ABCB1 in
the Endothelial cells (Gelati. M et al., 2005)
- in tumor vasculature and
brain stem cells
- regulation of ABCB1
reduction of tumor up to 90%
ABCC1 Brain Cancer (Bauer.B et al., 2008)
Related to the tumor
resistance.
Epilepsy (Sisodiya. S et al., 2002)
Astrocyes, neurons and
endothelial cells contain
of transporter ABCC1.
ABCC2 Brain Cancer (Bauer.B et al., 2008)
-Related to tumor resistance
in the endothelial cells.
- regulation in human
glioma, astrocytomas and
tumor vasculature.
Epilepsy (Ah. H et al., 2001)
- in endothelial cells from
epileptic patients
-Transports anti-epileptic
drugs such as phenytoin
ABCC4 Brain Cancer (leggas. M et al., 2004)
Associated with brain tumor
resistance
in astrocytic tumors

Epilepsy:

Although treatment of epilepsy is available, around 40% of patients fail to respond to the
medications. The cause of the drug resistance is not known and this has led to major concern,
however one hypothesis mentions that due to Epilepsy, the ABC drug efflux transporters are
over expressed specifically ABCB1. Tishler et al. made the seminal observation on patients
with drug-resistant epilepsy and showed that there was an increase in regulation of ABCB1
mRNA in the capillaries of the brain. After that test it has been discovered that the up
regulation took also place in astrocytes, microglia and neurons including the animal models
with epilepsy.[195]. Another study was done which was related to the inhibition of the up
regulation of ABCB1 caused by the epilepsy, results showed an increase of drug uptake.
However, the effect of the ABC transporters in AED-resistance epilepsy in still controversial
because it is not exactly known if the AEDs are actually substrates for ABC transporters,
especially ABCB1. (Sisodiya. S et al., 2002)

Multiple Sclerosis

local release of inflammatory cytokines and chemokines by activated leukocytes Resulting in

immune activation of endothelial cells, including the up regulation of cell adhesion molecule
(CAM) when there is loss in BBB and BCB which lead us to the need of additional immune
cells known as bystander leukocyte.
Studies provided evidence that BBB dysfunction is not the main leading cause to MS and
also found that it might precede immune cells infiltration, the infiltrated cells secrete cytokine
and chemokines that result in either the enhanced or decreased inflammatory processes.
(S.McQuaid,et al, 2009)
Overview:
The spinal cord and the brain which are protected by the myelin sheath are damaged during
MS which is classified as an autoimmune disease. The protective covering of the nerves gets
inflamed and damages the myelin sheath. There are two types of myelin damaging.
First when slight damage happens that cause a minor interruption in the nerve impulses.
Second type the damage is substantial therefore there will be complete disruption of nerve
impulses.
Signs and symptoms of MS can be unpredictable such as 1-Balance which is caused by the
affected coordination on the visual and spatial areas, 2-Memory problems caused by the
demyelination accompanied by MS, 3-Muscle weakness caused by the disruption of the
impulses that occurred either by inflamed or damaged nerves, 4-Uhthoffs phenomena (heat
intolerance) since there is demyelinated nerves accompanied with MS any raise or increase in
body temperature whether coming from physical part like exercise or emotional part like
anger it will lead to a delayed or blocked conduction of impulses.

Types of MS:
- Clinically isolated syndrome
- Relapsing-remitting MS
- Secondary-progressive MS
- Primary-progressive MS
- Progressive-relapsing MS

How to get MS is an unknown factor however the most privileged site is the CNS, now a
days certain studies showed that lack of endogenous antigen presenting cells, lack of
lymphatic system and presence of immunological BBB are the reasons of immune reactions
in the CNS.
Leukocytes importance:
There are three ways of entry of leukocytes into the CNS, the first route from the blood to the
CSF which has a physiological significance due to its content of 3000 per/ml leukocytes in
healthy individual and 80% of these leukocytes are T-cells, second route is from blood to
subarachnoid and then into the Virchow-robin which is significant due to immune
surveillance because its considered the site of lymphatic interaction with antigen presenting
cells, third route is the migration of the activated T-cells from blood to parenchymal
perivascular space (1).
(Ransohoff, et al. 2003)
BBB transport:

Immune responses are regulated by BBB mechanisms in case of MS and experimental

models of MS such as EAE. There are CD4 (T-lymphocytes) cells that are activated outside
the CNS in case of EAE.
Cells are then accumulate in the brain and CSF by passing two barriers which are the BBB
and the bloodCSF barrier .(Ransohoff, et al. 2003) .
Antigens presented by either subarachnoid spaces macrophages or pericytes are being
searched by CD4 T- lymphocytes which they carry a function of immune-surveillance of the
CNS.
There is a debate in the EAE models specifically the TH17 lymphocytes whether they play an
important role in neuronal killing (Stockinger, et al. 2007) or there is other cytotoxic T-
lymphocyte that cause neuronal killing. Its believed that TH17 secrete Interleukin17 and also
believed that these lymphocytes are activated in the peripheral organs (outside the CNS) then
they are transported across the BBB. The other part of the debate is either TH1, TH0 or PRE-
TH17 cross the BBB and get maturated (activated) inside the CNS.
The inflammation of post capillary venules on the pial surface determine the factors of
transport of leukocytes across the cerebral endothelium and these factors are: rolling,
activation, adhesion and transmigration (Engelhardt, et al. 2005)

In the rolling phase there is an interaction between selectins and their ligands, in the
activation phase there is interaction between chemokines and G-protein coupled receptors, in
adhesion phase there is interaction between integrins and endothelial cell adhesion molecule,
in transmigration phase there are three interactions between chemokines , chemokine
receptors and MMP.

Its required for interaction between G-protein-coupled receptor and their ligand (chemokine)
to mediate the activation of integrins to achieve adhesion. A second signal which connects the
luminal with abluminal sides of the BBB and G-protein-coupled receptor that results in
cytoskeletal reorganization which leads to transmigration of leukocytes across the BBB.
Extravasation of leukocytes by paracellular or transcellular route across the BBB can be done
by the participation of tight junction molecules including JAM-A and ESAM.
New therapies suggested transport of central memory T-cells, effector memory T-cells and
activated monocytes are blocked by the agent Natalizumab (Ransohoff, R.M. et al (2007).
T cells function on three ways firstly it expresses alfa4b1 integrins on the cell surface.
Secondly transportation from BBB to CSF. Thirdly their interference with MS lesions.
Natalizumab mechanism counteract the T-cells function by
- Binds and inactivate the integrins on the cell surface
- Reduction of BBB breakdown with active MS
- Lowering the counts of CSF leukocytes by 60% or more
AIDS Dementia:
ADC (AIDS Dementia Complex) is a late HIV1 infection but still its pathogensis unclear
which mostly complicate the CNS diseases and its highly related to morbidity and mortality
rates.
CNS pathology caused by HIV1, Encephalitis, Meningitis and other neuro-inflammatory
diseases is well developed when BBB cells are regulated crowdedly.
In case of HIV1 infection it infiltrate the CNS by specific mechanism which mediate the
glycoprotein GP120 which activates protein kinase C iso form in BEC to increase BBB
permeability, when its permeable it allows the migration of monocytes and macrophages
(Kanmogne, G.D., et al. 2007)
Studying the BBB molecular mechanisms is important for two things:
- Understanding the pathogenesis of the disease
- Lead to discovery of new therapeutic agents

For example the transport of monocytes carrying HIV1 virus into the BBB comes before the
AIDS Dementia, also the involvement of MMPS in neuronal diseases such as MS, HIV1,
Cerebral Ischemia and etc.
Alzheimers disease

Alzheimers disease in relation of tight junction

Alzheimers disease is characterized by decreased cognition function progressively.Studies

showed that it has a relation to ABC transporters as well as to tight junctions of the blood
brain barrier.

Many factors contribute in developing Alzheimer disease. First, increased levels of occludins
in astrocytes and neurons. Second, occludins accumulation reflects the BBB breakdown as a
result of chronic hypoxia and aberrant angiogenesis that leads to TJ protein surrouned by
cells.Third, accumulation of amyloid b-peptide(AB) in the brain which leads to accumulation
of neurotoxic oligomers of AB. Increased AB in neurovascular and vascular levels will result
in a condition called Cerebral Amyloid Angiopathy(CAA). Last onset of AD doesnt show
increase in AB in the brain cells but the false clearance of AB from the brain across BBB may
lead to accumulation of AB and formation of plaque in the blood vessels.

Alzheimers disease in relation of ABC transporters

Cells of the BBB, Neurovascular unit(NVU) and other brain cells detect many ABC
transporters families like A, B, C, D and G. Recently, studies show that some ABC families
like ABCB1, ABCC1 and ABCG2 have a role on the CNS. ABCA1 is the best described and
main transporter that contribute in brain homostasis. Dysfunction of ABCA1 leads to AD.
Three observations showed that relation between ABCA1 and AD. First, ABCA1 showed to
be apoE-dependent cholesterol efflux. Second, reducing apoElipidation leads to reduced A
breakdown and increased A. Third, in mice lacking ABCA1 decreased cholesterol clearance
leads to decreased apoElipidation.

Treatment approaches

Reduced clearance of Amyloid A peptide through A efflux leads to increase Amyloid A

peptide in the brain which is a leading cause of AD. Studies showed that up-regulation of
ABCB1 on the brain leads to clearance of Amyloid A peptide from brain. This is explained in
three observations. First, patient with increased level of A showed deacreased level of
ABCB1. Second, upregulation of ABCB1 on cells transports A. Third, in a transgenic model
of AD mouse, decreased ABCB1 function leads to accumulation of A in the brain. These
observations still under study if the up-regulation of ABCB1 could contribute to chronic
treatment of AD. Although the exact mechanisms still not known.

Recently, studies showed that in AD patient brain the ABCG4 mRNA and protein upregulated
in microgela which is located near to amyloid plaque. ABCG4 leads to cholesterol removal
from brain. Increased ABCG4 enhance apoElipidation which leads to A removal from the
brain. Accumulation of amyloid plaque leads to microglial respose associated with the
increase of ABCG4. This observation still under study if could be a part of AD therapy.

CNS Pathology Blood Brain Barrier Dysfunction

Trauma The Up-regulation of bradykinin leads to the stimulation of inflammatory

response leads to the production and release of interleukin-6 (IL-6) from
astrocytes, which leads to opening of the BBB.

HIV Causes disruption of the tight junctions in the blood brain barrier.

Parkinsons Disease The reduced efficacy of the P-glycoprotein leads to dysfunction of the BBB.
Brain tumors The decrease of tight junction protein claudin 3 and the redistribution of
astrocytes AQP4 and Kir4.1 lead to the breakdown of BBB.

Pain The alteration of the BBB tight junction expression and the BBB permeability
cause due to the inflammation.

Glaucoma Opening of the BBB, possibly through diffusion of endothelin-1

and matrix-metalloproteinase-9 into peri-capillary tissue.

Lysosomal storage Possible changes in the permeability of the BBB including the transport of it,
diseases (LSD) however it depends on the type of LSD

BBB structural alterations in the CNS. (N. Joan Abbott et al., 2010)
Conclusion

The BBB is one of the strongest barriers found in the human body against xenobiotics and
chemotherapeutics. This creates another barrier for the researchers to develop drug therapy to
treat diseases related to the brain such as brain cancer, MS, AZ and etc.

In fact, based on the research data done, it has been noticed that the BBB physiology during
the disease state changes and can lead to the increase of barrier against chemotherapeutics.
For example, during brain cancer ABC transporters are up-regulated which causes failure in
treating the condition. This also can be applied to epilepsy as ABC transporters are also up-
regulated. Other diseases mentioned in this research do also cause the increase of barrier due
to other physiological changes.

However, the researches regarding the treatment of the BBB in disease state is very limited.
Further studies must be done to identify the exact physiological changes and mechanisms
during the diseases state with the development of chemotherapeutic drugs with enhanced
efficacy related to the changes of the BBB disease conditions.
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